Genetic obesity in animal models is an invaluable tool for deciphering cause from effect in the complex series of behavioral, hormonal and metabolic changes that accompany disturbances in the regulation of body weight. Recent experiments from the applicant's laboratory have demonstrated that the first recognized obesity mutation, "the yellow mouse", is caused by ectopic expression of the agouti protein, a novel signaling molecule that normally controls the synthesis of pure yellow pigment in hair follicles. In mice that carry the lethal yellow (Ay) or the viable yellow (Avy) mutations, hyperphagia, hyperinsulinemia, and altered adipocyte metabolism are associated with expression of the agouti gene product in nearly every tissue of the body. However, the mechansim of agouti signaling in the hair follicle has not been determined, and it is not clear whether the initiating events in agouti-induced obesity occur at the level of the central nervous system, the pancreas, or the adipocyte. The biology and amino acid sequence of the agouti protein suggest that it acts locally in a paracrine fashion, and that proteolytic processing occurs during or shortly after secretion. As a first step towards understanding the normal agouti signaling pathway, antisera directed to specific regions of the agouti protein will be used to characterize its biosynthesis in vitro and to define its biologically active form in vivo. To determine if expression of agouti in a particular target tissue is either necessary or sufficient to cause obesity, transgenic mice will be constructed in which agouti expression is directed to the brain, to the integument, to the pancreas, or to adipocytes. At the cellular level, there are two hypotheses for agouti signaling. One postulates that agouti blocks the actions of alpha-melanocyte stimulating hormone (alpha-MSH) and related molecules at central nervous system receptors, in which case abnormalities in nutrient intake and the autonomic nervous system are likely to be primary events in the development of obesity. Alternatively, the agouti protein may bind to its own, as yet unidentified, receptor, in which case abnormalities in energy balance are likely to be primary events. These possibilities will be distinguished by determining the effects of agouti expression on melanocortin receptor binding and activation in cell culture. Finally, a potential role for agouti signaling in the normal regulation of body weight will be investigated by identifying and isolating agouti homologs in humans and in mice. The function of selected homologs in mice will be assessed in the whole animal by gene disruption in embryonic stem cells.